We have proposed a kinetic model for the actomyosin ATPase activity in which the myosin molecule oscillates back and forth between a state which binds strongly to actin and a state which binds weakly to actin. Relaxation occurs when the transition between the two states is blocked. In the present study, using myosin cross-linked to actin, we have developed further evidence that the rate-limiting step in the cycle is a special conformational change which occurs while the myosin is in the weak binding state. This conformational change follows the ATP hydrolysis step and occurs at about the same rate whether the myosin is bound to or dissociated from actin. We also obtained evidence that the weak binding of myosin.ATP to actin is a highly specific interaction. Both in the presence and absence of ATP, two isoenzymes of myosin show the same four-fold difference in their binding. Furthermore, the binding shows the same dependence on ionic strength in the presence and absence of ATP. This occurs,despite the fact that ATP weakens the binding more than four orders of magnitude. Finally, we have observed several phenomena predicted by our biochemical model in single skinned rabbit muscle fibers including the salt-dependent binding of myosin crossbridges to actin inrelaxed muscle.